Project Summary/Abstract This project is focused on the biology of telomeres, the protective repetitive sequences at chromosome ends whose erosion determines the replicative lifespan of primary human cells. Telomere dysfunction has been implicated in a number of human diseases, including inherited bone marrow failure syndromes and cancer. Our aim is to understand how telomeres protect chromosome ends and how they are maintained. We have gained insight into these issues through studies of shelterin, the six-subunit protein complex that specifically binds to telomeric DNA. Shelterin is anchored on telomeres by two double-stranded telomeric DNA binding proteins, TRF1 and TRF2, which promote telomere replication and protect telomeres from the DNA damage response, respectively. This proposal focuses on these two critical telomere factors. In AIM 1. we will investigate the function of TRF2. TRF2 is required for the formation of t-loops, the altered telomere structure formed by strand- invasion of the 3? telomeric overhang into the double-stranded telomeric DNA. The t-loop structure has been proposed to prevent activation of the ATM kinase by hiding the telomere terminus from the Double-strand Break (DSB) sensor (MRN) in the ATM pathway. Similarly, t-loops have been proposed to block the loading of Ku70/80 to prevent the c-NHEJ pathway from acting on chromosome ends. T-loop formation is thought to require the TRFH domain of TRF2 but its mode of action is unclear. We are analyzing the biochemical aspects of the TRFH domain and found novel features with likely relevance to t-loop formation that will be tested in vivo. In AIM 2, we will test the role of t-loops in telomere protection. We are developing orthogonal tools for TRF2-independent t- loop formation that will allow us to determine whether t-loops are sufficient to protect telomeres. In AIM 3, we will investigate the function of TRF1. Loss of TRF1 induces telomere replication problems manifesting as fragile telomeres, replication fork stalling, and sister telomere associations. Our data show how TRF1 uses the BLM helicase to prevent lagging-strand replication problems caused by G4 DNA. We propose to investigate how TRF1 prevents leading-strand replication problems, fork stalling, and sister telomere associations. We will expand on our unpublished data indicating that the sister telomere associations represent a novel type of telomere fusion resulting from a previously unrecognized alt-NHEJ mechanism involving stalled replication forks. In AIM 4, we will study the function of the Myb domains of TRF1 and TRF2. Our unpublished data challenge the widely-held view that Myb domains only function as DNA binding modules. We will pursue our hypothesis that the Myb domains of TRF1 and TRF2 mediate unanticipated protein-protein interactions with a critical roles in telomere protection and replication. The proposed experiments will use the biochemical, cell biological, genetic, and molecular tools for telomere analysis developed in our lab in combination with innovative experimental approaches. These experiments have the potential to lead to new concepts in telomere biology and are relevant to the role of telomeres in age-related human disease states.